Mitoxantrone and Mitoxantrone-Loaded Iron Oxide Nanoparticles Induce Cell Death in Human Pancreatic Ductal Adenocarcinoma Cell Spheroids

Pancreatic ductal adenocarcinoma is a hard-to-treat, deadly malignancy. Traditional treatments, such as surgery, radiation and chemotherapy, unfortunately are still not able to significantly improve long-term survival. Three-dimensional (3D) cell cultures might be a platform to study new drug types in a highly reproducible, resource-saving model within a relevant pathophysiological cellular microenvironment. We used a 3D culture of human pancreatic ductal adenocarcinoma cell lines to investigate a potential new treatment approach using superparamagnetic iron oxide nanoparticles (SPIONs) as a drug delivery system for mitoxantrone (MTO), a chemotherapeutic agent. We established a PaCa DD183 cell line and generated PANC-1SMAD4 (−/−) cells by using the CRISPR-Cas9 system, differing in a prognostically relevant mutation in the TGF-β pathway. Afterwards, we formed spheroids using PaCa DD183, PANC-1 and PANC-1SMAD4 (−/−) cells, and analyzed the uptake and cytotoxic effect of free MTO and MTO-loaded SPIONs by microscopy and flow cytometry. MTO and SPION–MTO-induced cell death in all tumor spheroids in a dose-dependent manner. Interestingly, spheroids with a SMAD4 mutation showed an increased uptake of MTO and SPION–MTO, while at the same time being more resistant to the cytotoxic effects of the chemotherapeutic agents. MTO-loaded SPIONs, with their ability for magnetic drug targeting, could be a future approach for treating pancreatic ductal adenocarcinomas.


Introduction
Pancreatic cancer is the seventh leading cause of death in the world, with an increasing incidence [1,2]. The most prevalent tumor type among pancreatic cancers is pancreatic ductal adenocarcinoma (PDAC), with an overall 5-years survival rate of 11%, and it is expected to become the second leading cause of cancer death by 2030 [3]. Its poor prognosis is strongly associated with the time point of diagnosis, especially when the carcinoma has already spread beyond the pancreas [4].
Recent genomic analyses identified 32 mutated genes in pancreatic adenocarcinoma, which are divided into four subgroups correlating with histopathological characteristics, namely immunogenic, pancreatic progenitor, squamous and aberrantly differentiated endocrine/exocrine tumors [5]. The genetic drivers of PDAC are predominantly mutations in the well-known cancer genes KRAS, CDKN2A, TP53 and SMAD4, besides other genes mutated at a lower prevalence [6]. SMAD4 mutations are correlated with worse clinical outcomes and these tumors are more prone to metastasize at a higher incidence in metastatic recurrence [7,8]. SMAD4 is capable of enabling gene transcription and tumor suppression via the TGF-β signaling pathway. Thus, it can control tumor development by facilitating growth arrest and an induction of apoptosis.
The poor survival rate (5-year survival < 11%) is not only due to the lack of early detection and delayed presentation caused by non-specific symptoms, but also because of inadequate therapies. Currently, surgical resection is the only treatment that offers a potential cure for pancreatic cancer. Supportive therapy through the application of chemotherapeutic agents may improve survival rates. There is some evidence that promises further improvement in survival with the administration of chemoradiotherapy in the neo-adjuvant setting [2,9]. Future treatment of this refractory disease may involve immunotherapy utilizing checkpoint inhibitors, protein, or whole-tumor-cell vaccines and nanoparticle-based drugs [9,10].
Nanoparticles, in particular superparamagnetic iron oxide nanoparticles (SPIONs), have become a new agent for treatment and diagnosis in cancer [11][12][13][14]. SPIONs can not only be used for imaging and for diagnostics of diseases, but also for enhancement of the accumulation and release of drugs at the pathological site, thereby increasing therapeutic efficacy and reducing the occurrence of side effects by decreasing their localization in healthy tissues [15,16]. Due to their magnetic properties, they can be controlled by an external magnetic field, allowing targeted delivery of therapeutics. This procedure, known as magnetic drug targeting (MDT), is characterized by high efficiency and effectiveness, as well as reduced side effects [17]. There are already several studies on the use of SPIONs for the diagnosis of pancreatic cancer using multimodal imaging, such as magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) [18,19]. In contrast, there is only a limited amount of literature on the therapy of pancreatic cancer using functionalized SPIONs. For instance, the effects of SPIONs coated with curcumin and multifunctionalized magnetic iron oxide nanoparticles containing an anti-CD47 antibody, as well as the chemotherapeutic agent, gemcitabine, have been the subjects of former research [20,21]. However, MTO, a cytostatic drug used for cancer therapy and multiple sclerosis treatment with a potency up to 20,000 times higher than that of gemcitabine, has shown promising preclinical results for the treatment of pancreatic cancer [22][23][24][25]. Nevertheless, MTO did not show a significant response rate in a phase II clinical study including patients with advanced pancreatic carcinoma, as one of the main therapeutic limitations were dose-dependent hematologic side effects [26]. Since MTO not only effectively disrupts DNA synthesis and DNA repair, but also induces immunogenic cell death, the multiple effects of MTO combined with the targeting capacity of SPIONs might be a future approach for the therapy of pancreatic ductal adenocarcinoma [27,28]. The still predominantly used in vitro method to develop new drugs or agents for cancer therapy is a two-dimensional (2D) cell culture. However, this does not reflect tumor biology adequately due to unlimited amounts of oxygen and nutrition, as well as unphysiological changes in cell morphology [29]. Experiments performed with 3D cell cultures provide more accurate data on tumor characteristics, cell-to-cell interactions, drug sensitivity and metabolic profiles, and are more similar to the complex microenvironment cells experience in vivo [29][30][31].
In this study, we generated 3D spheroids of three pancreatic ductal adenocarcinoma cell lines (PaCa DD183 from primary tumors, wild-type PANC-1 and PANC-1 SMAD4 (−/−) ) by seeding the trypsinized cells into an agarose-coated 96-well plate. Free MTO was tested in for its efficacy to reduce the viability of cells and growth of tumor spheroids depending on their SMAD4 mutation status. Furthermore, we investigated the efficacy of SPIONs loaded with MTO in comparison with its unbound counterpart. Interestingly, we demonstrated an increased uptake of MTO, as well as SPIONs loaded with MTO, in SMAD4-mutated spheroids. In contrast, SMAD4-mutated cells showed a significantly worse response rate to the effects of MTO and SPIONs bound MTO.

Synthesis of Superparamagnetic Iron Oxide Nanoparticles (SPIONs)
Lauric acid (LA)-coated SPIONs were synthesized according to the protocol of Zaloga et al., 2014 [32]. Briefly, Fe (II) chloride and Fe (III) chloride were dissolved in ultrapure water and co-precipitated by stirring at 80 • C in alkaline media, under an argon atmosphere. After coating with lauric acid, the suspension was homogenized for 30 min at 90 • C and dialyzed several times (MWCO of 10 kDa) with ultrapure water. Subsequent coating with a protein corona of human serum albumin (HSA) was performed according to the protocol of Zaloga et al., 2016 [33]. Briefly, HSA solution (Recombumin Elite, 10% w/v, Albumedix, Nottingham, UK) was sterilized using a 0.22 µm filter, transferred into dialysis bags (MWCO 10 kDa) and dialyzed against 4.5 L of ultrapure water (4 water changes, 5 h). After concentration by tangential flow ultrafiltration [34], the HSA solution was stirred at room temperature and lauric-acid-coated SPIONs were added dropwise through a 0.8 µm filter. Finally, excess HSA was removed by tangential flow ultrafiltration before the SPION solutions were filtered using a 0.22 µm sterile filter. MTO loading of SPIONs was performed as reported by Zaloga et al., 2016 [33]. Briefly, 900 µL of SPIONs (4.84 mg Fe/mL) was vortexed with 100 µL mitoxantrone solution (2 mg/mL) and incubated for 5 min, to obtain a SPION MTO

Iron Quantification of Nanoparticles
The iron content of the SPIONs was determined using an Agilent 4200 microwave plasma-atomic emission spectrometer (MP-AES) (Agilent Technologies, Santa Clara, CA, USA). The SPION suspension (50 µL) was dissolved in 50 µL 65% HNO 3 , and incubated for 10 min at 95 • C. Prior to analysis, the sample was further diluted in 1900 µL ultrapure water. A commercial iron solution was used as an external standard.

Dynamic Light Scattering (DLS) and Zeta Potential Measurements
A Malvern Zetasizer (Malvern Instruments, Worcestershire, UK) was used to determine the zeta potential and the hydrodynamic size of the particles in water. Particles were diluted to an absolute iron content of 50 µg/mL and measured in triplicate at 25 • C.

Generation of Pancreatic Carcinoma Spheroids
Cellular spheroids were produced in 96-well plates coated with 50 µL of 1.5% agarose. Briefly, cells were washed with PBS and detached from the cell culture surface using trypsin/EDTA. After dilution in 10 mL cell culture medium, the number of viable cells was analyzed by an automatic cell counter (MUSE ® Cell Analyzer, Merck-Millipore, Billerica, MA, USA). Agarose-coated wells were equipped with 150 µL medium and subsequently seeded with 50 µL (300,000 viable cells/mL) cell suspension, and incubated for 72 h at 37 • C.

Determination of Spheroid Growth by Transmission Microscopy
On day three, five and seven after seeding, the spheroid size was determined by analyzing microscopy images using an Axiovert 40 CFL Microscope and the Axio Vision SE64 Rel4.9 software (Zeiss, Jena, Germany). The areas of the 2D projections of the spheroids were determined by the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Experiments were conducted at least four times, with nine replicates each. Microsoft Excel was used for statistical analysis.

Hematoxylin/Eosin Staining of Spheroid Cryosections
Five representative spheroids from each cell line were selected for cryosection preparation. After removing the cell culture medium, the spheroids were carefully embedded in CryoGlue embedding medium. Twenty minutes later, spheroids were frozen at −20 • C for at least 24 h, before preparation of 10 µm sections using an MNT microtome (SLEE medical GmbH, Mainz, Germany). The sections were then transferred to slides until histological staining. For the staining, cryosections were washed with distilled water for 5 s before staining with Hematoxylin Gill III for 6 min. Subsequently, the sections were placed in 0.1% HCl for 5 s and rinsed with tap water for 3 min. Counterstaining was performed with 0.5% eosin for 6 min, followed by rinsing with tap water and embedding with DPX mounting medium. The sections were imaged and analyzed using an Axiovert 40 CFL Microscope and the Axio Vision SE64 Rel4.9 software (Zeiss, Jena, Germany).

Mitoxantrone Treatment of Spheroids
Seventy-two hours after seeding pancreatic cells in 96-well plates, spheroids were treated for an additional 4 days with various MTO concentrations (0, 0.5, 5, 10, 20 and 47.8 µg/mL) under standard cell culture conditions. Bright-field images were taken after three, five and seven days using an Axiovert 40 CFL Microscope and the Axio Vision SE64 Rel4.9 software (Zeiss, Jena, Germany). The areas of the 2D projections of the spheroids were determined by the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Experiments were conducted four times, with technical triplicates. Microsoft Excel was used for statistical analysis.

Treatment of Pancreatic Spheroids with SPION, SPION MTO and MTO
Pancreatic spheroids were treated with SPION (478 µg/mL Fe), SPION MTO (20 µg/mL) and free MTO (20 µg/mL) in the same MTO and iron concentration, 72 h after seeding. Spheroids were incubated for an additional 96 h under standard cell culture conditions, followed by imaging using a Zeiss microscope (Axio Observer Z.1, Zeiss, Jena, Germany).

Dissolving of Spheroids to Single-Cell Suspensions
Spheroids were harvested from 96-well plates and pooled in 15 mL cell culture flasks (Falcon, Sarstedt, Nümbrecht, Germany). The supernatant was removed, and the spheroids were washed with 500 µL PBS for at least 1 min. Afterward, the spheroids were incubated with 150 µL trypsin for 20-30 min. Spheroids were dissolved by repeated pipetting. Cell culture medium (750 µL) was added to neutralize the trypsin, and the suspension was centrifuged at 300× g for 5 min before carefully removing the supernatant. The resulting cell pellet was finally resuspended in 250 µL of medium.

Analysis of Viability and Cell Death Phenotype in Flow Cytometry
To determine viability, 50 µL of the single-cell suspensions were incubated with 250 µL of the staining mixture for 20 min at 4 • C. One milliliter of the staining solution contained 10 µg Hoechst 33342, 25 ng AxV-FITC and 66.6 ng PI per mL Ringer's solution. The fluorescence intensity was analyzed with a Gallios flow cytometer (Beckman Coulter, Fullerton, CA, USA). Excitation of FITC and PI was obtained at 488 nm; FITC fluorescence was verified with the FL1 sensor (525/38 nm band pass filter, BP); and the PI fluorescence with the FL3 sensor (620/30 nm BP). The MTO fluorescence was excited at 638 nm and recorded by the FL7 sensor (725/20 nm BP). Excitation of the Hoechst 33342 fluorescence was obtained at 405 nm and recorded by the FL9 sensor (430/40 nm BP). Electronic compensation was used to eliminate fluorescence bleed-through. Data analysis was conducted with the Kaluza software Version 1.2 (Beckman Coulter).

Physicochemical SPION Characterization
The physicochemical properties greatly influence not only the interaction between nanoparticles and cells, but also the final quality of the loaded particles. Therefore, the quality and reproducibility of particle synthesis was controlled by measurements of particle size and zeta potential using dynamic light scattering (DLS) ( Table 1). When freeze fracture transmission electron microscopy (TEM) was performed, we found that SPIONs formed multicore particles, of which the aggregate size was comparable to the size measured by dynamic light scattering (DLS). Every individual particle had a diameter of approximately 10 nm [33]. The zeta potential of SPIONs in ultrapure water showed a negative value of −21 mV. In water, the SPIONs exhibited hydrodynamic sizes of 65 nm. The absence of larger aggregates indicated high colloidal stability. In addition, previous studies demonstrated high colloidal stability of all particles in FCS-containing media, indicating the formation of a stabilizing protein corona and a successful binding of MTO to the particle surface [33,39]. In serum-containing medium and whole blood, even in the presence of a magnetic field, no permanent agglomerations were detected [40]. Thus, the physicochemical data suggest the suitability of the nanoparticles for subsequent in vitro experiments. MTO was freshly loaded onto the SPIONs before every experiment. Binding experiments indicated adsorption of 97.6 ± 0.1% of 500 µg MTO adsorbed to 1 mL of SPIONs (2 mg/mL) after 5 min equilibrium. The release of MTO from the particles incubated in RPMI-1640 medium was determined via dialysis and a magnetic assay setup. After 72 h, 11.6 ± 0.1% and 23.7 ± 0.4% were released from the former or the latter, respectively. Thus, MTO was released rather slowly from the particles, most likely by diffusion [33,39].

Generation of 3D Spheroids of Different PDAC Cell Lines
We used various PDAC cells lines to form spheroids. PaCa DD183 and PANC-1 originated from a primary PDAC [41]. Furthermore, the established cell line PANC-1 with intact SMAD4, as well with knocked-out SMAD4 (see Appendix A), was investigated. It is known that the SMAD4 gene is involved in gene regulation and tumor suppression via the TGF-β pathway. Furthermore, mutations are correlated with worse clinical outcomes and connected to higher metastasis rates [7,8].
After evaluating the optimal culture condition for each pancreatic ductal adenocarcinoma cell line, 15,000 cells were seeded for spheroid generation into agarose-coated 96-wellplates ( Figure 1). After spheroid structures were formed, spheroid size was determined microscopically after 3, 5 and 7 days of incubation (Figure 1a). Analysis of transmission microscopy images revealed large cell-line-dependent size differences during the initial phase of spheroid formation, with PANC-1 SMAD4 (−/−) producing the smallest and PANC-1 the largest spheroids (Figure 1c). In addition, the images and image analysis showed that between the third and seventh day, the size of all spheroids decreased significantly, indicating an increase in spheroid density [42]. After seven days, the edges of all spheroids, except those formed by PaCa DD183 cells, finally began to fray. Cryosections prepared on the third, fifth, and seventh day and subsequently stained with hematoxylin/eosin (HE) to visualize necrotic areas, revealed no evidence of necrotic cores (Figure 1b). This is consistent with previous reports, which demonstrated that mass transport in small 3D cell aggregates below 400-500 µm is sufficient to maintain cell viability through adequate diffusion of nutrients, oxygen and metabolic wastes [43][44][45].

Generation of 3D Spheroids of Different PDAC Cell Lines
We used various PDAC cells lines to form spheroids. PaCa DD183 and PANC-1 originated from a primary PDAC [41]. Furthermore, the established cell line PANC-1 with intact SMAD4, as well with knocked-out SMAD4 (see Appendix A), was investigated. It is known that the SMAD4 gene is involved in gene regulation and tumor suppression via the TGF-β pathway. Furthermore, mutations are correlated with worse clinical outcomes and connected to higher metastasis rates [7,8].
After evaluating the optimal culture condition for each pancreatic ductal adenocarcinoma cell line, 15,000 cells were seeded for spheroid generation into agarosecoated 96-well-plates (Figure 1). After spheroid structures were formed, spheroid size was determined microscopically after 3, 5 and 7 days of incubation (Figure 1a). Analysis of transmission microscopy images revealed large cell-line-dependent size differences during the initial phase of spheroid formation, with PANC-1 SMAD4 (−/−) producing the smallest and PANC-1 the largest spheroids (Figure 1c). In addition, the images and image analysis showed that between the third and seventh day, the size of all spheroids decreased significantly, indicating an increase in spheroid density [42]. After seven days, the edges of all spheroids, except those formed by PaCa DD183 cells, finally began to fray. Cryosections prepared on the third, fifth, and seventh day and subsequently stained with hematoxylin/eosin (HE) to visualize necrotic areas, revealed no evidence of necrotic cores (Figure 1b). This is consistent with previous reports, which demonstrated that mass transport in small 3D cell aggregates below 400-500 µm is sufficient to maintain cell viability through adequate diffusion of nutrients, oxygen and metabolic wastes [43][44][45].

Impact of Free MTO on PDAC Spheroid Growth
The effect of MTO treatment on the growth and viability of PDAC cells spheroids was examined after the addition of free MTO (0, 0.5, 5, 10, 20 and 47.8 µg/mL) on the third day of spheroid formation and during the following four days (Figure 2). Bright-field images on days 3, 5 and 7 confirmed the size reduction of untreated spheroids, as shown in Figure 1.
Compared with the size of untreated spheroids at days 5 and 7, treatment with 20 µg/mL and 47.8 µg/mL MTO resulted in a significant concentration-dependent increase in the size of PaCa DD183 and PANC-1 SMAD4 (−/−) spheroids, indicating an effect on cell cohesion at higher MTO concentrations (Figure 2a,b,e,f). Interestingly, the size of PANC-1 spheroids increased sharply at already 5 µg/mL and 10 µg/mL MTO, whereas the increase was less pronounced after incubation with 20 µg/mL and 47.8 g/mL MTO, indicating a very rapid cell death at MTO concentrations of 20 µg/mL or higher (Figure 2c,d).
The effect of MTO treatment on the growth and viability of PDAC cells spheroids was examined after the addition of free MTO (0, 0.5, 5, 10, 20 and 47.8 µg/mL) on the third day of spheroid formation and during the following four days (Figure 2). Bright-field images on days 3, 5 and 7 confirmed the size reduction of untreated spheroids, as shown in Figure 1. Growth progression was normalized to the spheroid size at day 3. Data are expressed as the mean with standard deviation (n = 4, with three replicates each). Statistical significances between MTOfree and MTO-treated spheroids are indicated with *, ** and ***. The respective confidential intervals are * p ≤ 0.05, ** p ≤ 0.0005 and *** p ≤ 3 × 10 −9 , respectively, and were calculated via the Student's ttest. Abbreviations: MTO, mitoxantrone. On day three, cell culture medium was supplemented with MTO (0, 0.5, 5, 10, 20 and 47.8 µg/mL, respectively). Growth progression was normalized to the spheroid size at day 3. Data are expressed as the mean with standard deviation (n = 4, with three replicates each). Statistical significances between MTO-free and MTO-treated spheroids are indicated with *, ** and ***. The respective confidential intervals are * p ≤ 0.05, ** p ≤ 0.0005 and *** p ≤ 3 × 10 −9 , respectively, and were calculated via the Student's t-test. Abbreviations: MTO, mitoxantrone.

Impact of Free MTO on Cell Viability within PDAC Spheroids
At day seven, we analyzed the viability of the spheroids via flow cytometry (Figure 3a,b) to get a clearer insight into the mechanism of death induced after 96 h incubation with MTO. As they are indicators of apoptosis and necrosis, we evaluated the cells for their phosphatidylserine exposure using Annexin V-FITC (Ax), and for their plasma membrane 2c,d).

Impact of Free MTO on Cell Viability within PDAC Spheroids
At day seven, we analyzed the viability of the spheroids via flow cytometry ( Figure  3a,b) to get a clearer insight into the mechanism of death induced after 96 h incubation with MTO. As they are indicators of apoptosis and necrosis, we evaluated the cells for their phosphatidylserine exposure using Annexin V-FITC (Ax), and for their plasma membrane integrity using propidium iodide (PI). Ax−PI are considered viable, Ax+PI apoptotic and PI+ cells necrotic.  The applied gating function, shown exemplarily for PaCa DD183 cells in Figure 3a, was used to analyze cell morphology, phosphatidylserine exposure and plasma membrane integrity and MTO uptake into the cells. PaCa DD183 cell spheroids revealed a dosedependent increase in apoptotic and necrotic cells with increasing MTO concentrations. At the highest MTO concentration (47.8 µg/mL), the number of viable cells decreased to 20.1 ± 6.7% (Figure 3b). In contrast to PaCa-DD183, the sensitivity of PANC-1 cell spheroids against MTO was much more pronounced. A concentration of 5 µg/mL reduced the viability to 2.2 ± 0.7%, compared to 70.4 ± 6.0% in PaCa DD183 cells. Thereby, the percentage of apoptotic cells increased to 92.4 ± 1.3%, while PaCa DD183 cells only 17.2 ± 2.5% apoptotic cells in the presence of 5 µL/mL MTO. Interestingly, PANC-1 cells with a mutated SMAD4 gene were much less sensitive to MTO compared to the original PANC-1 cells. Thus, the use of 5 µg/mL MTO resulted in only 33.4 ± 14.8% apoptotic and 2.8 ± 2.3% necrotic cells, and only at the highest MTO concentration (47.8 µg/mL), the cells reached approximately the same values that were already reached in the PANC-1 cells at 5 µg/mL.

Impact of Particle-Bound MTO and Free MTO on PDAC Spheroids
Based on the cytotoxicity data obtained with free MTO on PDAC spheroids, 20 µg/mL was selected as the MTO concentration for the comparison of effects achieved in free or nanoparticle-loaded form. Spheroids treated with non-loaded SPIONs (478 µg/mL Fe) or H 2 O served as the controls. After dissociation of spheroids into single cells, analysis of viability was performed by flow cytometry (Figure 4a-c). In PaCa DD183 cell spheroids, treatment with free MTO and SPION-linked MTO resulted in similar cytotoxicity with comparable numbers of viable cells (34.7 ± 1.8% and 38.8 ± 10.2%, respectively). There were also no significant differences in PANC-1 viable cells between treatment with free and bound MTO, although the effect on PANC-1 cells was much stronger (8.9 ± 3.2% and 3.6 ± 1.8%, respectively).

Discussion
PANC-1 SMAD4 (−/−) -derived spheroids similarly showed no significant differences in the number of viable cells after MTO or SPION MTO treatment (23.3 ± 0.6% and 16.0 ± 1.0%, respectively). However, the proportion of viable cells is more pronounced than in PANC-

Discussion
PANC-1 SMAD4 (−/−) -derived spheroids similarly showed no significant differences in the number of viable cells after MTO or SPION MTO treatment (23.3 ± 0.6% and 16.0 ± 1.0%, respectively). However, the proportion of viable cells is more pronounced than in PANC-1 SMAD4 (−/−) spheroids, confirming the decreased sensitivity to toxic substances in cells with downregulated or absent SMAD4 expression, as shown in Figure 3.
In PaCa DD183 and PANC-1 cells, we observed a decrease in the percentage of necrotic cells after SPION MTO -treatment compared to free MTO, which might be an indication for a slightly lower toxicity of SPION-bound MTO. However, this was not the case for PANC-1 SMAD4 (−/−) cells, which revealed slightly more necrosis when treated with SPION MTO in comparison to the free drug.
To find an explanation for the cell-specific differences in MTO effects, the cellular MTO amount was determined by analyzing the fluorescence intensity (Figure 4d-f). The increased cytotoxicity of MTO toward PANC-1 cells compared with PaCa DD183 cells correlated with an increase in cellular MTO concentration. In PANC-1 cells, fluorescence intensity upon treatment with free MTO or SPION MTO was 2.4-or 6.2-fold higher than in PaCa DD183 cells. Although the number of viable cells in PANC-1 SMAD4 (−/−) spheroids was higher than in spheroids derived from PANC-1 cells, the amount of MTO detected in PANC-1 SMAD4 (−/−) cells was 3.3-fold (free MTO) and 1.2-fold (SPION MTO ) higher than in PANC-1 cells. This indicates a higher uptake of free and particle-bound drug into the SMAD4-deletion mutant cells.
Since all solid tumors only occur as 3D structures in vivo, 3D cell culture systems may provide a way to bridge the gap between 2D cell cultures and the in vivo setting. In particular, analyzing the effects of drugs in cells grown in a monolayer cannot reflect the complexity of the 3D structure of a tumor [29]. Usually, in vivo studies are performed using animal models to overcome these problems. However, as the enormous number of mice and other animals used for research should be drastically reduced, in vitro testing systems need to be adapted to better approximate the real situation, which could lead to a reduction in animal testing, according to the 3Rs principle (replace-reduce-refine).
Testing pancreatic ductal adenocarcinoma presents a particular challenge, as this tumor entity is represented by hypoxic conditions and a dense specialized extracellular matrix/desmoplastic stroma within these tumors. Two-dimensional cell culture systems cannot replicate these features. Moreover, it has been shown that culturing human pancreatic adenocarcinoma cells in monolayers leads to a transition from epithelial cells to mesenchymal phenotypes [46]. Although it has already been shown by others that PDAC cell lines are able to form 3D spheroids, our results demonstrate that we could consistently reproduce tumor spheroids from PaCa DD183 and PANC-1 cell lines, as well as the newly generated SMAD4 knock-out cell line, PANC-1 SMAD4 (−/−) , to have a working platform for drug testing. Even though solid spheroids started to form after approximately 24 h, our goal was to obtain tightly packed tumor spheroids. Due to the 3D structure of the spheroid, the inner core has a limited amount of resources like oxygen and nutrients available, while metabolic waste needs to be transported out of the spheroid. Thus, the 3D model comes closer to reality than a 2D cell culture. One limitation of the used 3D spheroid model is the monoculture of PADC cells. In future studies this model can be further developed by coculturing cancerassociated fibroblasts and immune cells to improve resembling the characteristic dense stroma of PDACs [29,47].
After establishing highly reproducible tumor spheroids in PaCa DD183, PANC-1 and PANC-1 SMAD4 (−/−) tumor cell lines, we tested their resistance to mitoxantrone. In PaCa DD183 and PANC-1 SMAD4 (−/−) spheroids, MTO treatment resulted in a significant concentration-dependent increase in the area size (Figure 2a,b,e,f). Surprisingly, treatment of PANC-1 spheroids with MTO caused an increase in spheroid area at concentrations of 5 or 10 µg/mL MTO, while at lower and higher concentrations, the compact structure of the spheroids was not lost (Figure 2c,d). Whilst the non-dose-dependent behaviour is unusual at first, this might be explained by the fact that at concentrations of 5 and 10 µg/mL, apoptosis is massively induced in the spheroids, as detected by AxPI staining (Figure 4). Apoptosis is accompanied by the controlled shrinkage and condensation of the cell body and the degradation into multiple individual apoptotic bodies, which might explain the loss of coherence, and thus increase of area size. In contrast, MTO concentrations of 20 µg/mL might cause an immediate loss of plasma membrane integrity in PANC-1 spheroids due to an overload of toxic MTO, resulting in a higher percentage of necrotic cell death ( Figure 3). As this process is very quick, there might be no time for controlled degradation, and therefore a reduced loss of coherence.
Furthermore, our results demonstrate how genetically diverse PDAC tumor cell spheroids can respond differently to a given chemotherapeutic agent. PaCa DD183 and PANC-1 originated from a primary PDAC [41]. In the PANC-1 SMAD4 (−/−) cell clone, SMAD4 expression was knocked out using CRISPR/Cas9. It was previously pointed out that PANC-1 tumor spheroids are more resistant to chemotherapy and radiotherapy when harboring KRAS and p53 mutations compared to 2D cell cultures [48][49][50][51]. Therefore, we were interested in whether knocking out SMAD4 in the PANC-1 cell line, which has been found to be associated with a worse prognosis in pancreatic carcinoma, would affect resistance to MTO [52][53][54]. Interestingly, the uptake of free MTO and SPION bound MTO was increased in PANC-1 SMAD4 (−/−) cells. These findings correspond with the increased uptake of SPIONs in PANC-1 SMAD4 (−/−) cells in comparison to PANC1 cells, demonstrated by our research group and reported by Friedrich et al. [35]. Nevertheless, they showed a higher rate of viability compared to wildtype SMAD4 cells. Even though increased chemoresistance has been shown for other chemotherapeutic substances [55], to our knowledge this is the first study demonstrating an increased resistance to MTO in PANC-1 spheroids due to SMAD4 knockout.
We chose MTO and MTO-loaded SPIONs in this study because of their high chemotherapeutical potency and capabilities of inducing immunogenic cell death by the release of damage-associated molecular patterns. At the same time, MTO is quite stable, making it excellent for analysis even in complex media [22][23][24]56]. Our data point out that MTOloaded SPIONs, despite their slightly impaired penetration in comparison to unbound MTO, induce cell death reliably in pancreatic carcinoma spheroids.
Further research is needed to differentiate if MTO is released from the particles or if the particles penetrate into the spheroid together with the chemotherapeutic cargo. In previous investigations, with spheroids from HT-29 colon carcinoma cells, we observed a delayed penetration of MTO into the tumor spheroids when applied as SPION-MTO, by using fluorescence microscopy [57]. We hypothesize that the MTO must have been bound to the SPIONs at least initially, otherwise we would not have seen differences in uptake velocity and amount.
The cytotoxic potential of MTO comes along with severe dose-dependent side effects, which are the main therapeutical limitation for use in patients [26]. As demonstrated previously by our research group, MDT using MTO-loaded SPIONs could be a way to increase intratumoral cytotoxicity, while at the same time reducing systemic side effects [16,58,59]. It has been shown earlier that using an immunogenic cell death inductor conjugated on nanocarriers can induce a significant tumor reduction in pancreatic carcinomas [60]. Our group demonstrated previously that the loading of MTO onto SPIONs does not influence the capability of the drug to induce immunogenic cell death in tumor cells [27]. We also showed previously that the chemotherapeutic drug MTO can be accumulated in the tumor region, and the immune system was spared from the toxic effects of the drug [16]. The conservation of the immune system and the simultaneous induction of immunogenic cell death in the tumor region might improve therapeutic efficacy in PDAC.
Another possible future therapeutic application is the ability of SPIONs to locally induce hyperthermia by using alternating magnetic field-induced movement [61]. This could lead to an increased anti-tumoral activity, as it has been shown that mild hyperthermia can increase the cytotoxic effects of MTO [62,63].
With further research, combining the previously described features of SPIONs and MTO could open up possibilities for new, more effective treatment approaches regarding pancreatic carcinomas.

Conclusions
Our study demonstrated that 3D PDAC cell spheroids are a reproducible tool for in vitro assays. We found that nanoparticle-loaded MTO induced cell death in 3D PDAC cell spheroids. Furthermore, knocking out SMAD4 in PANC-1 cell spheroids led to an increased uptake of unbound and bound MTO. Interestingly, we demonstrated an increased cell resistance to the cytotoxic effects of free and bound MTO. This highlights the importance of the SMAD4 mutation for future treatment attempts. Potentially, a deeper understanding of genetic key mutations such as SMAD4 could enable an individualized treatment with an improved clinical outcome. Further research is needed to evaluate SPION potential for MDT in the field of pancreatic carcinomas. Locally increasing the intratumoral drug concentration, as well as reducing systemic side effects via MDT, could be a future approach to enhance the therapeutic options for patients with pancreatic carcinomas.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data are available on reasonable request from the corresponding author. study-Panc1. We performed a genetic knock-out of this gene by the CRISPR-Cas9 system [64]. Seventy-two hours after transfection, the positive cells were extracted from the culture by FACS-sorting due to the green GFP signal, and seeded into 96-well plates for single clone selection. These single clones were evaluated for successful knock-out of the SMAD4 gene by Western blot. For clones 1 and 2 originating from knock-out by the sgRNA1 pair, complete homologous absence of SMAD4 could be shown, while the sgRNA1 pair seemed to produce a truncated form of SMAD4. We therefore selected clone sgRNA1-1 (PANC-1 SG1-SC1) for performing further experiments.